US10610576B2 - Precision glycoconjugates as therapeutic tools - Google Patents

Precision glycoconjugates as therapeutic tools Download PDF

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US10610576B2
US10610576B2 US16/362,363 US201916362363A US10610576B2 US 10610576 B2 US10610576 B2 US 10610576B2 US 201916362363 A US201916362363 A US 201916362363A US 10610576 B2 US10610576 B2 US 10610576B2
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carrier protein
thiol
carbohydrate antigen
carbohydrate
glycoconjugate
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US20190290746A1 (en
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Tze Chieh SHIAO
Rene Roy
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Koranex Capital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/001172—Sialyl-Thomson-nouvelle antigen [sTn]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/04—Disaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the present description relates to glycoconjugate therapeutic tools. More specifically, the present description relates to carbohydrate antigens non-randomly coupled to free thiol groups of immunogenic and antigenic carrier peptides and proteins, and improved methods of producing same using, for example, photocatalytic thiol-ene “click chemistry” reactions. Applications as antigens, immunogens, vaccines, and in diagnostics are also described.
  • the ultimate objective of immunotherapy is to trigger the innate and adaptive responses of the immune system in a way similar to that produced during an infection or tumor progression.
  • the principal interfaces between the innate and adaptive immune responses are the antigen-presenting cells (APCs), and particularly dendritic cells (DCs).
  • APCs are able to recognize microorganisms through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs).
  • PRRs pattern recognition receptors
  • TLRs Toll-like receptors
  • the microorganisms or tumors and their related antigenic markers can be engulfed by the APC through an endocytic pathway where it is typically degraded.
  • the peptides and covalently linked antigens released by protein processing are then displayed on MHC class II molecules and are recognized by CD4+ T cells which in turn undergo functional maturation into different subsets, such as Th1 or Th2 cells, upon co-stimulatory signals received from the APC.
  • Th1 cells lead to a predominantly pro-inflammatory response with the secretion of IFN- ⁇ and TNF- ⁇ , whereas Th2 cells secrete typical cytokines.
  • Th1 cells are mainly associated with a cell-mediated response
  • both types of Th cells support the production of antibodies by B cells, which in turn influences antibody isotype and function.
  • IL-12 and TNF- ⁇ are associated with the differentiation of Th1 cells and production of type 1 IgG subclasses
  • IL-6 and other Th2 cytokines contribute to the type 2 IgG subclass (IgG1) production. It is thus desirable to be able to tailor vaccine-induced immunity to an appropriate response to deal with a pathogen or tumor antigen of interest.
  • Carbohydrates as opposed to proteins and peptides, are T cell independent antigens not properly equipped to trigger the participation of Th cells and hence, cannot induce immune cell proliferation, antibody class switching, and affinity/specificity maturation.
  • the major early advances initially encountered with carbohydrate-based vaccines have been supported by the discovery that, when properly conjugated to protein carriers, serving as T cell dependent epitopes, bacterial capsular polysaccharides became capable of acquiring the requisite immunochemical ability to produce opsonophagocytic antibodies.
  • glycoconjugate vaccines are inadequate and face significant regulatory and/or commercial obstacles, since the preparations lack the necessary homogeneity in terms of their carbohydrate distribution and reproducibility (i.e., the attachment points of the sugars onto the proteins are randomly distributed and in various densities from batch to batch).
  • glycoconjugate vaccines having greater carbohydrate antigen homogeneity, more precisely characterizable structures, and reproducibility from batch to batch would be highly desirable.
  • the present description relates to glycoconjugate immunogens comprising carbohydrate antigens directly coupled to immunogenic carrier proteins at precise (non-random) positions. More particularly, the carrier proteins comprise one or more free thiol groups (e.g., corresponding to the side chains of cysteine residues) and the carbohydrate antigens are conjugated to the carrier proteins at one or more of these free thiol groups.
  • the present description also relates to improved methods for synthesizing glycoconjugate immunogens/vaccines involving directly conjugating carbohydrate antigens to free thiol groups of carrier proteins, for example using “click-chemistry” approaches (e.g., photocatalytic thiol-ene reactions).
  • the improved conjugation methods described herein may be performed under conditions sufficiently mild (e.g., use of only water-soluble reagents, the absence of organic solvents, or use concentrations of organic solvents sufficiently low (e.g., ⁇ 5%) to avoid carrier protein denaturation, and/or at relatively neutral pH) to avoid destroying the activity, antigenicity, and/or structure (e.g., cleavage of native disulfide bridges and/or denaturation) of a carrier protein, without affecting the specificity of the conjugation.
  • conditions sufficiently mild e.g., use of only water-soluble reagents, the absence of organic solvents, or use concentrations of organic solvents sufficiently low (e.g., ⁇ 5%) to avoid carrier protein denaturation, and/or at relatively neutral pH) to avoid destroying the activity, antigenicity, and/or structure (e.g., cleavage of native disulfide bridges and/or denaturation) of a carrier protein, without affecting the specificity of the conjugation.
  • photocatalytic thiol-ene reactions described herein may be performed under ultraviolet light in the presence of a catalyst (e.g., 355 nm or 365 nm), or under short-wave ultraviolet light (e.g., at 254 nm) in the absence of a catalyst, further simplifying the process.
  • a catalyst e.g., 355 nm or 365 nm
  • short-wave ultraviolet light e.g., at 254 nm
  • the present description relates to a method for producing a glycoconjugate immunogen, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal alkene (alkenyl carbohydrate antigen), the terminal alkene being directly conjugatable to a thiol group via a thiol-ene reaction; (b) providing a carrier protein having one or more free thiol groups; and (c) performing a photocatalytic thiol-ene reaction to directly conjugate the carbohydrate antigen to the carrier protein at the one or more free thiol groups, thereby producing the glycoconjugate immunogen; wherein the carrier protein is immunogenic when administered to a subject, and wherein conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen upon administration to the subject, as compared to administration of the unconjugated carbohydrate antigen.
  • the photocatalytic thiol-ene reaction is performed under reaction conditions that avoid carrier protein denaturation.
  • the alkenyl carbohydrate antigen is water-soluble and the photocatalytic thiol-ene reaction is performed under reaction conditions that retain the carrier protein's activity, antigenicity, and/or structure (e.g., in the absence of any organic solvent, or in the presence of an organic solvent at a concentration sufficiently low (e.g., ⁇ 5%) to avoid carrier protein denaturation).
  • the photocatalytic thiol-ene reaction may be performed in the presence of a catalyst (e.g., a water-soluble or water-insoluble catalyst) under irradiation under short-wave (e.g., 254 nm) and/or long-wave (e.g., 355 or 365 nm) ultraviolet light.
  • a catalyst e.g., a water-soluble or water-insoluble catalyst
  • short-wave e.g., 254 nm
  • long-wave e.g., 355 or 365 nm
  • the photocatalytic thiol-ene reaction comprises reacting between 1 to 200 molar equivalents of the alkenyl carbohydrate antigen per free thiol group of the carrier protein; and/or the photocatalytic thiol-ene reaction is performed at a pH between about 3 and 10, 3.5 and 9.5, 4 and 9, 4.5 and 8.5, 5 and 8, 5.5 and 8, 6 and 8, or 6.5 and 7.5.
  • the carbohydrate antigen following conjugation to the carrier protein, is not cleavable from the carrier protein by an endogenous enzyme of the subject.
  • the alkenyl carbohydrate antigen is covalently linked to the terminal alkene, and/or the carbohydrate antigen is conjugated to the carrier protein, via a glycosidic bond, such as is an O-glycosidic bond, an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination, such as between an allyl amine and a reducing sugar (including bacterial CPS).
  • a glycosidic bond such as is an O-glycosidic bond, an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination, such as between an allyl amine and a reducing
  • the carbohydrate antigen comprises a B cell epitope, and/or induces a humoral immune response in the subject. In some embodiments, the carbohydrate antigen comprises a T cell epitope, and/or induces a cell-mediated immune response in the subject. In some embodiments, the carbohydrate antigen comprises both a B cell epitope and a T cell epitope, and/or induces both a humoral and a cell-mediated immune response in the subject.
  • the carbohydrate antigen is or comprises a tumor associated carbohydrate antigen (TACA), such as Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex, or any combination thereof.
  • TACA tumor associated carbohydrate antigen
  • the photocatalytic thiol-ene reaction conjugates at least two of the same carbohydrate antigen or more than one type of carbohydrate antigen to the carrier protein, thereby producing a multi-valent glycoconjugate immunogen (e.g., comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the same or different types of carbohydrate antigens conjugated to the carrier protein).
  • a multi-valent glycoconjugate immunogen e.g., comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the same or different types of carbohydrate antigens conjugated to the carrier protein.
  • the carbohydrate antigen is or comprises a viral polysaccharide antigen, or a bacterial capsular polysaccharide (CPS) (e.g., a Pneumococcal and/or Streptococcal polysaccharide serotype, meningococcal CPS; influenza (such as influenza type a or b) CPS).
  • CPS bacterial capsular polysaccharide
  • the alkenyl carbohydrate antigen is linked to the terminal alkene via a linker (e.g., a linker as described herein).
  • a linker e.g., a linker as described herein.
  • the carrier protein comprises one or more cysteine residues having the one or more free thiol groups. In some embodiments, the carrier protein comprises a T cell epitope, and/or induces a cell-mediated immune response in the subject.
  • the carrier protein comprises a B cell epitope, and/or induces a humoral immune response in the subject. In some embodiments, the carrier protein comprises both a B cell epitope and a T cell epitope, and/or induces both a humoral and a cell-mediated immune response in the subject. In some embodiments, the carrier protein is, is from, or comprises: Tetanus Toxoid (TT), Diphtheria Toxoid (DT), cross-reacting material 197 (CRM197), Meningococcal Outer Membrane Protein Complex (OMPC), H.
  • TT Tetanus Toxoid
  • DT Diphtheria Toxoid
  • CCM197 cross-reacting material 197
  • OMPC Meningococcal Outer Membrane Protein Complex
  • the carrier protein is a protein having one or more disulfide bridges, and wherein: (i) the one or more disulfide bridges remain unaffected following said photocatalytic thiol-ene reaction; or (ii) the carrier protein is pre-treated with a reducing agent to expose one or more additional free thiol groups for conjugation to the carbohydrate antigen.
  • the total number of carbohydrate antigens comprised in the glycoconjugate immunogen is equal to the number of free thiol groups available on the carrier protein prior to conjugation.
  • the glycoconjugate immunogen induces a cell-mediated immune response to the carbohydrate antigen upon administration to the subject.
  • the methods described herein further comprise: (d) purifying the glycoconjugate immunogen.
  • the present description relates to a method for producing a glycoconjugate vaccine, the method comprising formulating the glycoconjugate immunogen prepared by a method described herein with a pharmaceutically acceptable excipient, and/or an adjuvant (e.g., an inorganic compound, a mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, a toll-like receptor agonist, an immunostimulatory polynucleotide (e.g., CPG), an immunostimulatory lipid, Freund's adjuvant, RIBI's adjuvant, QS-21, muramyl dipeptide, or any combination thereof).
  • an adjuvant e.g., an inorganic compound, a mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent-accessible cysteine residues, wherein the one or more carbohydrate antigens are linked to the immunogenic carrier protein at the one or more solvent-accessible cysteine residues, and wherein conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens upon administration to a subject, as compared to administration of the unconjugated carbohydrate antigen.
  • the one or more carbohydrate antigens are linked to the one or more solvent-accessible cysteine residues via a linker as described herein and the one or more carbohydrate antigens is/are as described herein.
  • the synthetic glycoconjugate immunogen is a multi-valent glycoconjugate immunogen as described herein, and uses a carrier protein as described herein.
  • the total number of carbohydrate antigens comprised in the glycoconjugate immunogen is equal to the number of solvent-accessible cysteine residues on the carrier protein.
  • the synthetic glycoconjugate immunogen induces a cell-mediated immune response to the carbohydrate antigen upon administration to the subject.
  • the synthetic glycoconjugate immunogen is prepared by a method described herein.
  • the present description relates to a glycoconjugate vaccine produced by a method as described herein, and/or comprising the synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient, and/or an adjuvant.
  • the glycoconjugate vaccine is a prophylactic vaccine or a therapeutic vaccine.
  • the present description relates to a method of immunizing, vaccinating, or treating a subject comprising administering to the subject the glycoconjugate immunogen produced by a method as described herein, the glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a glycoconjugate vaccine as described herein.
  • the present description relates to a synthetic glycoconjugate immunogen or a glycoconjugate vaccine as described herein, for use in immunizing, vaccinating, or treating a subject having a disease, or for detecting the presence of an antibody that specifically binds to the glycoconjugate, or for detecting said immunization, vaccination, or treatment (e.g., in a biological sample from a subject).
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as designed above, wherein y is an integer varying from 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the structure:
  • SA is a sugar antigen or a portion thereof;
  • S—PC is a carrier protein;
  • X is O, S, NR 1 , or CH 2 ;
  • R 1 is —H, —COH, —COCH 3 , or —COEt;
  • n is 1, 2, 3, 4, or 5; and
  • R 2 is H or Me; or a stereoisomer thereof.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the structure:
  • SA is a sugar antigen or a portion thereof;
  • S—PC is a carrier protein;
  • X is S, NR 1 , CH 2 or O;
  • R 1 is —H, —COH, —COMe, or —COEt;
  • n is 1, 2, 3, 4, or 5;
  • R 2 is H or Me;
  • q is 1, 2, 3, 4, or 5;
  • R 3 and R 4 are each a hydrogen atom and m is 1, 2, 3, 4 or 5, or R 3 and R 4 form together a radical —CO—CH 2 — or a radical —CO—CH 2 —CH 2 — with the carbonyl linked to the nitrogen atom, and m is 1; or a stereoisomer thereof.
  • the sugar antigen is a carbohydrate antigen as described herein
  • the carrier protein is as described herein
  • the synthetic glycoconjugate immunogen is a multi-valent glycoconjugate immunogen as described herein.
  • the present description relates to a vaccine comprising the synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient and/or an adjuvant as described herein.
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, or a synthetic glycoconjugate immunogen as described herein, for the manufacture of a vaccine.
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, a glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein, for the treatment of a subject having a disease associated with increased expression of said one or more carbohydrate or sugar antigens.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • protein e.g., in the expression “carrier protein” means any peptide-linked chain of amino acids, which may or may not comprise any type of modification (e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.), so long as the modifications do not destroy the immunogenicity of the glycoconjugate immunogens and glycoconjugate vaccines described herein.
  • modification e.g., chemical or post-translational modifications such as acetylation, phosphorylation, glycosylation, sulfation, sumoylation, prenylation, ubiquitination, etc.
  • administration may comprise administration routes such as parenteral (e.g., subcutaneously, intradermally, intramuscularly, or intravenously), oral, transdermal, intranasal, etc., so long as the route of administration results in the generation of an immune response in the subject.
  • parenteral e.g., subcutaneously, intradermally, intramuscularly, or intravenously
  • oral transdermal, intranasal, etc.
  • subject generally refers to a mammal, including primates, and particularly to a human.
  • FIG. 1 shows examples of typical chemical structures of key tumor-associated carbohydrate antigens (TACAs) from human glycoproteins (MUCs).
  • TACAs tumor-associated carbohydrate antigens
  • MUCs human glycoproteins
  • FIG. 2 shows a proposed “click chemistry” approach for conjugating a carbohydrate antigen (B cell epitope) directly to an available thiol group of an immunogenic carrier protein (e.g., T cell epitope such as Tetanus Toxoid).
  • an immunogenic carrier protein e.g., T cell epitope such as Tetanus Toxoid
  • FIG. 3 shows an example of a “precision glycoconjugate” prepared by a photocatalytic thiol-ene reaction to conjugate Tn or TF antigens to six native “free” cysteine residues on the carrier protein Tetanus Toxoid.
  • FIG. 4 shows an example of a “precision glycoconjugate” prepared by using Tn or TF antigens linked to thiol-specific iodoacetamido or maleimido groups, which are then conjugated to “free” cysteine residues on a carrier protein via a reaction a pH below 8 (iodoacetamido or maleimido groups) or a thiol-ene reaction (maleimido group).
  • FIG. 5 shows reaction schemes for the syntheses of allyl Tn antigen and allyl TF antigen ready for conjugation to carrier proteins, as described in Examples 2-6.
  • FIG. 6A-6D shows the 1 H-NMR ( FIG. 6A ) and 13 C-NMR ( FIG. 6B ) spectra, as well as mass spectrometry results ( FIGS. 6C and 6D ) for allyl 2-acetamido-3,6-di-O-pivaloyl-2-deoxy- ⁇ -D-glucopyranoside (Compound 2).
  • FIG. 7A-7D shows the 1 H-NMR ( FIG. 7A ) and 13 C-NMR ( FIG. 7B ) spectra, as well as mass spectrometry results ( FIGS. 7C and 7D ) for allyl 2-acetamido-2-deoxy- ⁇ -D-galactopyranoside (allyl Tn).
  • FIG. 8A-8C shows the 1 H-NMR ( FIG. 8A ) spectra, as well as mass spectrometry results ( FIGS. 8B & 8C ) for allyl 2-acetamido-4,6-O-benzylidene-2-deoxy- ⁇ -D-galactopyranoside (Compound 4).
  • FIG. 9A-9D shows the 1 H-NMR ( FIG. 9A ) and 13 C-NMR ( FIG. 9B ) spectra, as well as mass spectrometry results ( FIGS. 9C and 9D ) for allyl (2,3,4,6-tetra-O-benzoyl- ⁇ -D-galactopyranosyl)-(1 ⁇ 3)-2-acetamido-4,6-O-benzylidene-2-deoxy- ⁇ -D-galactopyranoside (Compound 6)
  • FIG. 10A-10D shows the 1 H-NMR ( FIG. 10A ) and 13 C-NMR ( FIG. 10B ) spectra, as well as mass spectrometry results ( FIGS. 10C and 10D ) for Compound 7.
  • FIG. 11A-11D shows the 1 H-NMR ( FIG. 11A ) and 13 C-NMR ( FIG. 11B ) spectra, as well as mass spectrometry results ( FIGS. 11C and 11D ) for allyl ( ⁇ -D-galactopyranosyl)-(1 ⁇ 3)-2-acetamido-2-deoxy- ⁇ -D-galactopyranoside (allyl TF).
  • FIG. 12 shows the results of Dynamic Light Scattering (DLS) analyses of the TF-TT glycoconjugate alone (upper left panel), peanut lectin alone (upper right panel), or both combined (lower panel).
  • DLS Dynamic Light Scattering
  • FIG. 13 shows the thiol concentration of the peptide dTT831-840 as a function of time in the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn and O-Allyl-TF.
  • FIG. 14 shows the fold reduction of thiols of the peptide dTT831-840 in the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn as a function of the peptide's cysteine position and the presence of AAPH or DMPA.
  • FIG. 15 shows the reactivity of lectins to peptide dTT831-840 conjugated to Tn by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn as a function of the position of the peptide's cysteine and the presence of AAPH or DMPA.
  • FIG. 16 shows the kinetics of thiol concentration of the peptide dTT831-840 in the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn at different wave lengths in the presence of AAPH or DMPA.
  • FIG. 17 shows the reactivity of the lectin Vicia Villosa to the peptide dTT831-840 conjugated to Tn by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn at different wave lengths in the presence of AAPH or DMPA.
  • FIG. 18 shows the kinetics of thiol concentration of the peptide dTT831-840 in the photocatalytic thiol-ene conjugation reaction with C-Allyl and O-Allyl sugars.
  • FIG. 19 shows the reactivity of lectins and anti-TF antibody to peptide dTT831-840 conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn.
  • FIG. 20 shows the reactivity of anti-TF antibody to reduced dTT or reduced dTT conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-TF.
  • FIG. 21 shows the molar ratio of thiols of dTT at various steps of the photocatalytic thiol-ene conjugation reaction and following reduction of the conjugated dTT.
  • FIG. 22 shows the reactivity to lectins and anti-TF mAb of dTT conjugated by the photocatalytic thiol-ene conjugation reaction to O-Allyl-Tn.
  • FIG. 23 shows the molar ratio of thiol of chemically thiolated dTT conjugated or not by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn in the presence of low or high amount of AAPH.
  • FIG. 24 shows the reactivity of the lectin Vicia Villosa to chemically thiolated dTT conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn in the presence of low or high amount of AAPH at two different times.
  • FIG. 25 shows the reactivity of lectins and anti-TF antibody to reduced BSA conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn.
  • FIG. 26 shows the relationship between the reducing conditions and the molar ratio of thiols of reduced BSA.
  • FIG. 27 shows the reactivity of the Tn-specific Vicia Villosa lectin and anti-TF antibody to BSA with various free thiol molar ratio and conjugated by the photocatalytic thiol-ene conjugation reaction with various amount of O-Allyl-Tn.
  • FIG. 28 shows the relationship between the reducing conditions, the molar ratio of thiols of reduced BSA and the TF reactivity of reduced BSA conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-TF.
  • FIG. 29 shows the molar ratio of thiols adducts in BSA in function of time by chemical thiolation alone or thiolation with simultaneous photocatalytic thiol-ene conjugation reaction in the presence or absence of AAPH and O-Allyl-Tn.
  • FIG. 30 shows the reactivity of the Tn-specific Vicia Villosa lectin with thiolated BSA or BSA simultaneously thiolated and conjugated by photocatalytic thiol-ene conjugation reaction in the presence or absence of AAPH and O-Allyl-Tn.
  • FIG. 31 shows the relationship between the thiol molar ratio and the reactivity of the Tn-specific Vicia Villosa lectin of BSA conjugated by the photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn in a function of time.
  • FIG. 32 shows the reactivity of the Tn-specific Vicia Villosa lectin to the native or thiolated BSA conjugated by photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn in the presence of low or high amount of AAPH and at two wave-lengths.
  • FIG. 33 shows the relationship between the galactose molar ratio and the reactivity of the anti-TF mAb to BSA at different steps of thiolation and photocatalytic thiol-ene conjugation reaction with O-Allyl-Tn at low or high amount of AAPH.
  • FIG. 34 shows the chemical structures of the dTT831-844-Cys- ⁇ Ala and the Cys-dTT831-844- ⁇ Ala with a cysteine residue at the C- and N-terminal, respectively together with their tabulated LC-MS data.
  • FIG. 35 shows the detailed LC-MS data of the peptide dTT831-844-Cys- ⁇ Ala.
  • FIG. 36 shows the detailed LC-MS data of the peptide Cys-dTT831-844- ⁇ Ala.
  • FIG. 37 shows the photolytic AAPH-catalyzed thiol-ene reaction of the O-Allyl Tn on the C-terminal peptide dTT831-844-Cys- ⁇ Ala.
  • FIG. 38 shows the LC-MS profile of the O-Allyl Tn on the C-terminal peptide
  • FIG. 39 shows the photolytic AAPH-catalyzed thiol-ene reaction of the O-Allyl Tn on the N-terminal peptide Cys-dTT831-844- ⁇ Ala.
  • FIG. 40 shows the LC-MS profile of the O-Allyl Tn on the N-terminal peptide.
  • glycoconjugate immunogens e.g., for use as vaccines, in diagnostics, or for generating diagnostic or therapeutic tools such as specific anti-glycoconjugate antibodies.
  • the glycoconjugate immunogens described herein generally comprise carbohydrate antigens coupled to immunogenic carrier proteins. More particularly, the carrier proteins comprise one or more free thiol groups (e.g., corresponding to the side chains of cysteine residues) and the carbohydrate antigens are conjugated to the carrier proteins at one or more of these free thiol groups.
  • the present description also relates to improved methods for synthesizing glycoconjugate immunogens/vaccines involving directly conjugating carbohydrate antigens to free thiol groups of carrier proteins, for example using “click-chemistry” approaches (e.g., photocatalytic thiol-ene reactions).
  • the improved conjugation methods described herein may be performed under conditions sufficiently mild (e.g., use of only water-soluble reagents, the absence of organic solvents, use concentrations of organic solvents sufficiently low (e.g., ⁇ 5%) to avoid carrier protein denaturation, and/or at relatively neutral pH) to avoid destroying the activity, antigenicity, and/or structure (e.g., cleavage of native disulfide bridges and/or denaturation) of a carrier protein, without affecting the specificity of the conjugation.
  • conditions sufficiently mild e.g., use of only water-soluble reagents, the absence of organic solvents, use concentrations of organic solvents sufficiently low (e.g., ⁇ 5%) to avoid carrier protein denaturation, and/or at relatively neutral pH) to avoid destroying the activity, antigenicity, and/or structure (e.g., cleavage of native disulfide bridges and/or denaturation) of a carrier protein, without affecting the specificity of the conjugation.
  • photocatalytic thiol-ene reactions described herein may be performed under ultraviolet light in the presence of a catalyst (e.g., 355 nm or 365 nm), or under short-wave ultraviolet light (e.g., at 254 nm) in the absence of a catalyst, further simplifying the process.
  • a catalyst e.g., 355 nm or 365 nm
  • short-wave ultraviolet light e.g., at 254 nm
  • the glycoconjugate immunogens/vaccines described herein may have greater homogeneity (in terms of their carbohydrate distributions), reproducibility, and may be easier to characterize as compared to glycoconjugates that rely on random coupling to other amino acid side chains (e.g., amines from lysine, or acid from glutamate/aspartate residues). Such characteristics may facilitate regulatory approval and/or commercialization of glycoconjugate vaccines, both of which have historically proven difficult based on traditional approaches.
  • the present description relates to a method for producing a glycoconjugate immunogen (e.g., for administration to a subject).
  • a subject generally refers to a living being (e.g., animal or human) that is able to mount an immune response to a glycoconjugate immunogen as described herein, preferably leading to the production of antibodies that specifically bind to the glycoconjugate immunogen.
  • a subject described herein may be a patient to be treated therapeutically (e.g., via vaccination with a glycoconjugate immunogen described herein) or may be employed as a means for generating tools (e.g., antibodies) for research, diagnostic, and/or therapeutic purposes.
  • the method for producing a glycoconjugate immunogen generally comprises providing a carbohydrate antigen having (e.g., chemically modified to comprise) a thiol-specific functional group; providing a carrier protein having one or more free thiol groups (e.g., one or more solvent-accessible cysteine residues, and/or cysteines not involved in disulfide bridges); and reacting the carbohydrate antigen with the carrier protein, thereby coupling of the carbohydrate antigen to the carrier protein at one or more predictable (non-random) attachment points corresponding to the positions of the free thiol groups of the carrier protein.
  • the method may further comprise purifying or isolating the glycoconjugate immunogen, which may then be formulated as a vaccine (e.g., comprising an adjuvant).
  • glycoconjugate refers to a carbohydrate antigen (e.g., an antigenic monosaccharide, di-saccharide, oligo-saccharide, or polysaccharide) coupled to a carrier protein in order to enhance the immunogenicity of carbohydrate antigen in a subject of interest.
  • carbohydrate antigen e.g., an antigenic monosaccharide, di-saccharide, oligo-saccharide, or polysaccharide
  • carrier protein in order to enhance the immunogenicity of carbohydrate antigen in a subject of interest.
  • the expressions “carbohydrate antigen” and “sugar antigen” carry the same meaning as used herein.
  • immunogen refers to an agent that is capable of being specifically bound by components of the immune system (e.g., by an antibody and/or lymphocytes), and generating a humoral and/or cell-mediated immune response in a subject of interest.
  • the term “immunogen” in an expression such as “glycoconjugate immunogen” refers to the ability (i.e., physical characteristic or property) of the glycoconjugate without limiting the glycoconjugate itself to a particular use (e.g., as an immunogen for generating an immune response in a subject).
  • a glycoconjugate immunogen described herein may be employed in diagnostic assays or methods (e.g., in vitro methods) to detect the presence or absence of an antibody that binds to the glycoconjugate immunogen in a biological sample (e.g., from a subject).
  • the glycoconjugate immunogens described herein may be used for screening, identifying, or evaluating antibodies that bind specifically to the glycoconjugate immunogen (e.g., monoclonal antibodies that are diagnostically or therapeutically applicable).
  • the present description relates to the use of a “click chemistry-type” approach for directly conjugating carbohydrate antigens to free thiol groups of carrier proteins. More specifically, as shown in FIG. 2 , photocatalytic thiol-ene reactions are preferred in which carbohydrate antigens (R 2 ) are modified to contain a terminal alkene functionality (alkenyl carbohydrate antigen) suitable for the direct covalent attachment to the cysteine thiol groups (—SH) of carrier proteins (R 1 ) using a photocatalytic thiol reaction.
  • —SH cysteine thiol groups
  • a carbohydrate antigen comprising a B-cell epitope may be photochemically conjugated via a thiol-ene reaction to an immunogenic carrier protein such as tetanus toxoid (TT), which is known to have six free thiol groups owing to its six available cysteine residues (excluding four cysteine residues involved in disulfide bridges).
  • TT tetanus toxoid
  • Conjugation methods described herein in the case of the tetanus toxoid carrier protein shown in FIG. 3 , preferably result in precisely six carbohydrate antigens being conjugated at predictable (non-random) attachment points.
  • the methods described herein are applicable to any mono-, oligo-, and polysaccharides, natural or synthetic, that can be made to end with an alkene group (terminal alkene).
  • the carbohydrate antigens described herein may be chemically modified to be linked (directly or indirectly via a linker or spacer) to a terminal alkene (e.g., via a glycosidic bond or a bond obtained by reductive amination, such as between an allyl amine and a reducing sugar, preferably using NaBH 4 and/or NaBH 3 CN), wherein the terminal alkene group of the alkenyl carbohydrate antigen is conjugatable to a free thiol of a carrier protein (e.g., one or more solvent-accessible cysteine residues, and/or cysteines not involved in disulfide bridges) via a thiol-ene reaction (e.g., a photocatalytic thiol-ene reaction).
  • a carrier protein e.g., one or more solvent-accessible cysteine residues, and/or cysteines not involved in disulfide bridges
  • a thiol-ene reaction e
  • the terminal alkene group of the alkenyl carbohydrate antigen may be a monosubstituted alkene, a vinyl group, or an allyl group.
  • the alkenyl carbohydrate antigen is water-soluble, enabling its use in aqueous-based thiol-ene reactions described herein.
  • the “glycosidic bond” may comprise one or more of an S-glycosidic bond, an N-glycosidic bond, an O-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination of a reducing sugar (e.g., using NaBH 4 or preferably NaBH 3 CN).
  • the glycosidic bond may be one that is not cleavable by an endogenous enzyme (e.g., a glycohydrolase) of the subject to be administered.
  • the glycosidic bond may be an S-glycosidic bond, an N-glycosidic bond, or a C-glycosidic bond, or a bond obtained by reductive amination, such as between an allyl amine and a reducing sugar.
  • the use of a “linker” or “spacer” is preferred and such terms are used herein to refer to a chemical linkage that provides sufficient physical separation of the carbohydrate antigen from the carrier protein to which it is conjugated to allow the carbohydrate antigen to be recognized by the immune system of a subject (e.g., as opposed to being masked by the carrier protein).
  • the linker may comprise a chain of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous residues selected from C, S, N, and O.
  • conjugate refers to the ability or capability of at least two molecules (e.g., a carbohydrate antigen and a carrier protein) to be covalently bonded to one another via a chemical reaction, regardless of whether the molecules are actually covalently bonded to one another.
  • conjuggated refers to at least two molecules (e.g., a carbohydrate antigen and a carrier protein) which are covalently bonded to one another.
  • carbohydrate antigens may be made to end with a terminal alkene group that is directly conjugatable to a thiol group via a thiol-ene reaction.
  • carbohydrate antigens having a terminal alkene group may be synthesized by adapting the approaches described herein in Examples 2-7 for the synthesis of the allyl Tn and allyl TF reactants.
  • the carbohydrate antigens having a terminal alkene group may be conjugated to free thiol groups of carrier proteins using photocatalytic thiol-ene reactions, for example, by adapting the approaches described herein in Examples 8 and 9 for the synthesis of the Tn-TT and the TF-TT conjugates.
  • an aqueous solvent e.g., a buffer such as PBS
  • a carrier protein also dissolved in an aqueous solvent (e.g., a buffer such as PBS) in a vessel (e.g., quartz cell) suitable for ultraviolet light irradiation.
  • the mixture is then irradiated under short-wave, medium-wave or long-wave ultraviolet light (e.g., having a peak wavelength at about 254 nm, at about 355 nm, or at about 365 nm), in the presence or absence of a catalyst (e.g., a photoinitiator or activator).
  • a catalyst e.g., a photoinitiator or activator.
  • the terms “catalyst,” “photoinitiator,” and “activator” may be used interchangeably to refer to substances that accelerate conjugation of a carbohydrate antigen to a carrier protein at the one or more free thiol groups via a photocatalytic thiol-ene reaction.
  • the catalyst may be a water-soluble photoinitiator such as a water-soluble free radical-generating azo compound; 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (Vazo 44 or VA-044); 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH); metals or metal ions having photoinitiator activity; or any derivative thereof having photoinitiator activity in a photocatalytic thiol-ene reaction described herein.
  • a water-soluble photoinitiator such as a water-soluble free radical-generating azo compound; 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (Vazo 44 or VA-044); 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH); metals or metal ions having photoinitiator activity; or any derivative thereof having photoinitiator activity in a
  • the catalyst may be a water-soluble photoinitiator such as a water-soluble peroxide such as tert-butyl hydroperoxide or benzoylperoxide, or ammonium persulfate, or other suitable catalysts.
  • a water-soluble photoinitiator such as a water-soluble peroxide such as tert-butyl hydroperoxide or benzoylperoxide, or ammonium persulfate, or other suitable catalysts.
  • the catalyst may be a water-insoluble photoinitiator such as a water-insoluble free radical-generating azo compound; 2,2-dimethoxy-2-phenylacetophenone (DMPA), azobisisobutyronitrile (AIBN), 2,2′-azobis(2-methylpropionitrile), 4,4′-azobis(4-cyanopentanoic acid) (ACVA), 1,1′-Azobis(cyanocyclohexane) (ACHN), diazenedicarboxylic acid bis(N,N-dimethylamide) (TMAD); azodicarboxylic acid dipiperidide (ADD), or any derivative thereof having photoinitiator activity in a photocatalytic thiol-ene reaction described herein.
  • DMPA 2,2-dimethoxy-2-phenylacetophenone
  • AIBN azobisisobutyronitrile
  • 2-methylpropionitrile 2,2′-azobis(2-methylpropionitrile
  • ACVA 1,1′-Az
  • the photocatalytic thiol-ene reactions described herein may comprise controlling the ratio of molar equivalents of the alkenyl carbohydrate antigen (i.e., carbohydrate antigen comprising a terminal alkene) per free thiol group of the carrier protein, for example, to reduce, minimize, or avoid sugar polymerization.
  • the photocatalytic thiol-ene reactions described herein may comprise reacting between 1 to 300, 1 to 250, 1 to 200, 1 to 100, or 1 to 10, 1 to 5, or 1 to 2 molar equivalents of the carbohydrate antigen per free thiol group of the carrier protein.
  • the photocatalytic thiol-ene reactions described herein are performed for sufficient time to achieve at least a 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50-fold reduction in total free thiol concentration in the carrier protein (e.g., as determined by Ellman test). In some embodiments, the photocatalytic thiol-ene reactions described herein are performed for 10 to 300, 10 to 270, 10 to 240, 10 to 210, 10 to 180, 10 to 150, 10 to 120, 10 to 90, 10 to 60, or 10 to 30 minutes.
  • the photocatalytic thiol-ene reactions described herein may be performed at a pH between about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, and about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.
  • the photocatalytic thiol-ene reactions described herein may be performed at a pH that avoids carrier protein denaturation.
  • the photocatalytic thiol-ene reactions described herein may be performed at a pH of between 4 and 5, preferably at pH 4.4 (e.g., in an acetate buffer).
  • the use of a water-soluble photoinitiator may be advantageous, as the thiol-ene conjugation reactions described herein may be performed only using aqueous reagents and aqueous solvents, since organic solvents may contribute to denaturation of the carrier protein, as well as undesired/unpredictable conjugation to non-free cysteine residues (e.g., cysteines involved in intramolecular and/or intermolecular disulfide bonds), as described in Dondoni et al., 2009 and Dondoni et al., 2012.
  • a water-insoluble photoinitiator may be employed together with an organic solvent, as needed, for dissolution thereof.
  • the presence or concentration of the organic solvent shall not contribute to denaturation of the carrier protein, as well as undesired/unpredictable conjugation to non-free cysteine residues (e.g., cysteines involved in intramolecular and/or intermolecular disulfide bonds).
  • such a photoinitiator may be one such as 2,2-dimethoxy-2-phenylacetophenone (DMPA).
  • the carbohydrate antigens described herein may be modified to have one or more thiol-specific functional groups such as iodoacetamides, maleimides, benzylic halides, or bromomethylketones, which react by S-alkylation of thiols to generate stable thioether products (e.g., carried out at relatively low pHs such as below 9, 8, 7.5, or 7, and above 5, 5.5, or 6, which may avoid unwanted, random carbohydrate antigen conjugations to lysine residues).
  • thiol-specific functional groups such as iodoacetamides, maleimides, benzylic halides, or bromomethylketones
  • carbohydrate antigens that have been modified to comprise thiol-specific functional groups (e.g., iodoacetamido and maleimido groups) are shown in FIG. 4 .
  • the maleimido groups qualify as terminal alkenes that are conjugatable to thiol groups of carrier proteins via a thiol-ene reaction.
  • the carbohydrate antigens described herein may comprise one or more B cell epitopes, and/or may induce a humoral immune response, and/or may comprise a T cell epitope, and/or induce a cell-mediated immune response in a subject upon administration.
  • glycoconjugate immunogens described herein may induce at least a cell-mediated immune response (e.g., in addition to a humoral response) to the carbohydrate antigen upon administration to a subject.
  • the carbohydrate antigens described herein may be or comprise, for example, a tumor associated carbohydrate antigen (TACA).
  • TACA tumor associated carbohydrate antigen
  • Glycoproteins and glycolipids of the outer cell membranes of cancer cells overexpress particular O-glycans.
  • These glycoproteins constitute a family of proteins collectively known as mucins (MUCs) with MUC1 representing the most widely investigated.
  • MUCs on normal cells are heavily O-glycosylated due to active glycosyltransferases which give rise to complex glycosylation patterns.
  • down-regulation of key glycosyltransferases trigger the accumulation of shorter glycans, giving rise to much more limited O-glycosylation on mucins.
  • TACAs which are otherwise cryptic (masked) by the complex glycosylation on normal tissues.
  • TACAs include the Tn antigen (a monosaccharide having the structure N-acetylgalactosamine (GalNAc)), the TF antigen (Thomsen-Friedenreich antigen, a disaccharide having the structure Gal ⁇ 1-3GalNAc ⁇ 1), and their cognate sialylated analogs, respectively.
  • the TACA described herein is, is from, or comprises: Tn antigen, STn antigen, Thomsen-Friedenreich (TF) antigen, (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex, or any combination thereof.
  • Tn antigen STn antigen
  • TF Thomsen-Friedenreich
  • TF Thomsen-Friedenreich
  • Globo H GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, sLex, or any combination thereof.
  • the structures of some common TACAs are shown in FIG. 1 .
  • the expression “is from”, when used in the context of carbohydrate antigens, refers to carbohydrate variants derived from a known carbohydrate antigen, wherein the variant retains at least the antigenicity of the known carbohydrate antigen.
  • carbohydrate antigens refers to carbohydrate variants derived from a known carbohydrate antigen, wherein the variant retains at least the antigenicity of the known carbohydrate antigen.
  • the synthesis of alkenyl-ending TACAs can be performed by both chemical as well as by chemoenzymatic processes, as described in Danishefsky et al., 2015.
  • the carbohydrate antigens described herein may be or comprise carbohydrate antigens associated with infectious agents, such as, but not limited to, bacteria and/or viruses, or associated with infections, such as, but not limited to bacterial infections and/or viral infections.
  • the carbohydrate antigens described herein may be or comprise, for example, a viral polysaccharide antigen, or a bacterial capsular polysaccharide (CPS).
  • the bacterial CPS is, is from, or comprises a Pneumococcal and/or Streptococcal polysaccharide serotype, meningococcal CPS, or influenza (such as influenza type a and b) CPS.
  • the conjugation methods described herein may conjugate the same or more than one type of carbohydrate antigen to the carrier protein, thereby producing a multi-valent glycoconjugate immunogen.
  • the multi-valent glycoconjugate immunogens described herein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the same or different types of carbohydrate antigens conjugated to a carrier protein.
  • the multi-valent glycoconjugate immunogens described herein may comprise more than one carbohydrate antigen that is conjugated to a single free thiol group on the carrier protein (e.g., via branched linker).
  • the multi-valent glycoconjugate immunogens described herein may comprise a plurality (e.g., at least 3, 4, 5, 6, 7 8, 9. 10, 11, 12, 13, 14, 15, or more) of carbohydrate antigens that are conjugated to a single free thiol group on the carrier protein as a dendrimer (e.g., via linkers having extensive branching).
  • the carrier proteins described herein comprise one or more free thiol groups.
  • free thiol or “free thiol group” refers to carrier proteins having one or more sulfhydryl groups that are available for chemical modification and/or conjugation (e.g., to a carbohydrate antigen as described herein).
  • the free thiol concentration of a given carrier protein may be measured for example using the Ellman test using a cysteine standard curve, as described in the present Examples.
  • the free thiol concentration of a given carrier protein may be expressed as fold reduction in free thiol concentration.
  • the glycoconjugates described herein may have at least a 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, or 2-fold decrease in free thiol concentration following a conjugation method described herein (e.g., as measured by Ellman test).
  • the carrier proteins described herein may be engineered to add one or more further cysteine residues, for example at the amino terminus, the carboxy terminus, or any solvent-accessible position of the carrier protein therebetween (as opposed to at positions that are not solvent accessible or “buried” within the three-dimensional structure of the protein carrier).
  • free thiol groups of protein carriers described herein may be defined as cysteines that are readily conjugatable to an alkenyl carbohydrate antigen by a photocatalytic thiol-ene reaction, when the reaction is performed under conditions sufficiently mild to not destroy the immunogenicity, structure, or activity of the carrier protein.
  • the term “activity” as used herein in relation to a carrier protein refers to the ability of the carrier protein to preserve the immunogenicity of the carbohydrate antigen (e.g., to an antibody that is known to specifically bind to the carbohydrate antigen).
  • the free thiol group may refer to one or more solvent-accessible cysteine residues of the carrier protein, and/or cysteines that are not involved in disulfide bridges, which in some cases may be important to maintain the structure of the carrier protein.
  • the carrier protein may be a protein having one or more disulfide bridges, and wherein the one or more disulfide bridges remain unaffected (i.e., intact) following conjugation to the carbohydrate antigen.
  • the carrier proteins described herein do not comprise free thiol-groups at their N and/or C-termini.
  • the carrier proteins described herein may comprise, or be engineered to comprise, a free thiol-group at their N and/or C-termini.
  • the carrier protein may be a protein having one or more disulfide bridges, and wherein the carrier protein may be pre-treated with a reducing agent (e.g., dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethylamine-HCl) to expose one or more additional free thiol groups for conjugation to the carbohydrate antigen.
  • a reducing agent e.g., dithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethylamine-HCl
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • the carrier protein prior to or together with performing the photocatalytic thiol-ene reaction, may be pre-treated with a reducing agent (e.g., as described herein). This pre-treatment may expose additional free thiol groups available for conjugation.
  • the carrier protein, prior to or together with performing the photocatalytic thiol-ene reaction may be pre-treated with a thiolating agent (e.g., 2-imminothiolane, N-hydroxysuccinimide dithiopropionate (DPS)). This pre-treatment may be employed to increase the number free thiol groups available for conjugation.
  • the carrier protein, prior to or together with performing the photocatalytic thiol-ene reaction may be pre-treated with a thiolating agent, and subsequently pre-treated with a reducing agent.
  • the carrier protein may preferably lack a cysteine-rich domain (e.g., a segment of at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids comprising at least 50% of cysteine residues).
  • a cysteine-rich domain e.g., a segment of at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids comprising at least 50% of cysteine residues.
  • the carrier protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 total cysteine residues. In some embodiments, the carrier protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 total free cysteine residues.
  • the total number of carbohydrate antigens comprised in the glycoconjugate immunogens described herein is equal to the number of free thiol groups available on the carrier protein prior to conjugation.
  • the glycoconjugate immunogens described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbohydrate antigens per carrier protein.
  • the present description relates to a composition
  • a composition comprising glycoconjugate immunogens having about or at least 70%, 75%, 80%, 85%, 90%, or 95% homogeneity in terms of carbohydrate conjugation species (e.g., at least 90% of glycoconjugate immunogen species/molecules in the composition have the same number of carbohydrate antigens conjugated to the carrier protein).
  • the carrier proteins described herein are proteins (e.g., peptides or polypeptides) that are preferably immunogenic to a subject that is to be administered to the glycoconjugate immunogen.
  • conjugation of the carbohydrate antigen to the carrier protein increases the immunogenicity of the carbohydrate antigen upon administration to the subject, as compared to administration of the unconjugated carbohydrate antigen (i.e., the carbohydrate antigen administered to the subject alone).
  • the carrier proteins may comprise a T cell epitope, and/or induce a cell-mediated immune response in the subject upon administration.
  • the carrier protein is a protein that is exogenous to the subject to be administered, which preferably has no (close) ortholog in the subject.
  • a carrier protein described herein refers to a “carrier protein suitable for human use” or simply “suitable carrier protein”, which means a carrier protein that is antigenically distinct from human proteins such that the carrier protein would not be considered as a “self-antigen” in humans.
  • suitable carrier protein a carrier protein that is antigenically distinct from human proteins such that the carrier protein would not be considered as a “self-antigen” in humans.
  • the use of carrier proteins that are too antigenically similar to corresponding human proteins may result in the carrier protein being considered as a “self-antigen”, which may not be ideal in human vaccines.
  • glycoconjugate immunogens consisting of TF antigen randomly conjugated to the ⁇ -amino groups of lysine residues of bovine serum albumin (BSA) have been previously described and characterized (e.g., Demian et al., 2014; Rittenhouse-Diakun et al., 1998; Heimburg et al., 2006; Tati et al., 2017).
  • BSA bovine serum albumin
  • the carrier protein is not albumin (e.g., bovine serum albumin).
  • the carrier protein described herein may be a protein that has already received regulatory (e.g., FDA) approval for administration to human subjects (e.g., in approved vaccines).
  • the carrier protein is, is from, or comprises Tetanus Toxoid (TT), Diphtheria Toxoid (DT), cross-reacting material 197 (CRM197), Meningococcal Outer Membrane Protein Complex (OMPC), H. influenzae Protein D (HiD), a cytokine, or an immunogenic fragment (or variant) thereof.
  • the carrier protein may be TT which contains 10 cysteine residues, 4 of which being engaged in disulfide bridges, giving rise to a glycoconjugate immunogen having 6 conjugated carbohydrate antigens.
  • the carrier protein may be CRM197 which contains 4 cysteine residues, giving rise to a glycoconjugate immunogen having 4 conjugated carbohydrate antigens.
  • the carrier proteins may by engineered to introduce additional cysteine residues in order to introduce additional free thiol groups for conjugation to carbohydrate antigens.
  • the cysteine residues may be engineered at solvent exposed portions of the carrier protein.
  • the present description relates to a method for producing a glycoconjugate vaccine or an immune response-triggering composition.
  • the method may comprise formulating the glycoconjugate immunogen described herein with a pharmaceutically acceptable excipient, and/or an adjuvant.
  • the adjuvant is or comprises: an inorganic compound, a mineral oil, a microbial derivative, a plant derivative, a cytokine, squalene, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, a toll-like receptor agonist, an immunostimulatory polynucleotide (e.g., CPG), an immunostimulatory lipid, Freund's adjuvant, RIBI's adjuvant, QS-21, muramyl dipeptide, TiterMaxTM, SteviuneTM, StimuneTM, or any combination thereof.
  • an immunostimulatory polynucleotide e.g., CPG
  • an immunostimulatory lipid e.g., Freund's adjuvant, RIBI's adjuvant, QS-21, muramyl dipeptide, TiterMaxTM, SteviuneTM, StimuneTM, or any combination thereof.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more carbohydrate antigens and an immunogenic carrier protein having one or more solvent-accessible cysteine residues, wherein the one or more carbohydrate antigens are linked to the immunogenic carrier protein at the one or more solvent-accessible cysteine residues.
  • the conjugation of the one or more carbohydrate antigens to the immunogenic carrier protein increases the immunogenicity of the one or more carbohydrate antigens upon administration to a subject, as compared to administration of the unconjugated carbohydrate antigen.
  • synthetic refers to a compound that is not a product of nature, which is produced by human intervention.
  • glycoconjugate immunogens described herein may comprise more than one species of carbohydrate antigen (e.g., more than one type of TACA) conjugated to the same carrier protein.
  • glycoconjugate immunogens described herein may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more types or species of carbohydrate antigen (e.g., TACA).
  • glycoconjugate immunogens described herein may comprise any combination of TACAs selected from Tn, S-Tn, Thomsen-Friedenreich (TF), (2,3)-S-TF, (2,6)-S-TF, Globo H, GD2, GD3, GM2, GM3, N-glycolyl-GM3, Lea, sLea, Lex, and sLex.
  • ratio in the combination of each TACAs may vary with the targeted tumor and may comprise between 1 to 20 molar ratios. In this way, the glycoconjugate immunogens described herein may be tailored, for example, to specific forms of cancer that are associated with increased expression of particular combinations of multiple TACAs.
  • the present description relates to a glycoconjugate vaccine which is produced by a method described herein, and/or which comprises a synthetic glycoconjugate immunogen described herein, and further comprises a pharmaceutically acceptable excipient, and/or an adjuvant.
  • the term “vaccine” refers to a composition comprising a glycoconjugate immunogen described herein that is administered a subject to provide a therapeutic benefit to the subject.
  • the glycoconjugate vaccines described herein may be a prophylactic vaccine or a therapeutic vaccine.
  • Vaccine compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration.
  • the route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, ocular) or via a parenteral route (e.g., intradermal, intramuscular, subcutaneous).
  • Vaccine compositions can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies.
  • Forms of administration may include suspensions and preparations for parenteral, subcutaneous, intradermal or intramuscular administration (e.g., injectable administration) such as sterile suspensions or emulsions.
  • Vaccines may be administered as a spray or mixed in food and/or water or delivered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like.
  • a suitable carrier diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard pharmaceutical texts, such as “Remington's Pharmaceutical Sciences,” 1990 may be consulted to prepare suitable preparations, without undue experimentation.
  • the present description relates to a method of immunizing, vaccinating, or treating a subject comprising administering to the subject a glycoconjugate immunogen or a glycoconjugate vaccine described herein.
  • the present description relates to a carbohydrate antigen chemically modified to be linked to a sulfhydryl-specific (thiol specific) functional group.
  • the carbohydrate antigen is chemically modified to be linked to a terminal alkene (e.g., via a glycosidic bond), wherein the terminal alkene group is conjugatable to a free thiol of a carrier protein via a thiol-ene reaction.
  • the carbohydrate antigen and the terminal alkene group are linked via a linker as described herein.
  • the terminal alkene group may be a vinyl group or an allyl group.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the structure:
  • SA is a sugar antigen or a portion thereof;
  • S—PC is a carrier protein;
  • X is O, S, NR 1 , or CH 2 ;
  • R 1 is —H, —COH, —COCH 3 , or —COEt;
  • n is 1, 2, 3, 4, or 5; and
  • R 2 is H or Me; or a stereoisomer (e.g., diastereomer) thereof.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein each linker has the structure:
  • SA is a sugar antigen or a portion thereof;
  • S—PC is a carrier protein;
  • X is S, NR 1 , CH 2 or O;
  • R 1 is —H, —COH, —COMe, or —COEt;
  • n is 1, 2, 3, 4, or 5;
  • R 2 is H or Me;
  • q is 1, 2, 3, 4, or 5;
  • R 3 and R 4 are each a hydrogen atom and m is 1, 2, 3, 4 or 5, or R 3 and R 4 form together a radical —CO—CH 2 — or a radical —CO—CH 2 —CH 2 — with the carbonyl linked to the nitrogen atom, and m is 1; or a stereoisomer (e.g., diastereomer) thereof.
  • X is O
  • the sugar antigen does not comprise Tn or STn.
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as defined above, wherein the linker has the structure:
  • the present description relates to the synthetic glycoconjugate immunogen as described above, wherein y is an integer varying from 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10, 1 to 5, or 1 to 2.
  • the sugar antigen may be a carbohydrate antigen as described herein, and/or the carrier protein may be a carrier protein as described herein.
  • the synthetic glycoconjugate immunogen may be a multi-valent glycoconjugate immunogen, for example, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the same or different types of carbohydrate antigens conjugated to a carrier protein.
  • the glycoconjugate immunogen may induce a cell-mediated immune response to the carbohydrate antigen upon administration to a subject.
  • the present description relates to a vaccine comprising a synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient and/or an adjuvant (e.g., an adjuvant as described herein).
  • a vaccine comprising a synthetic glycoconjugate immunogen as described herein, and a pharmaceutically acceptable excipient and/or an adjuvant (e.g., an adjuvant as described herein).
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, or a synthetic glycoconjugate immunogen as described herein, for the manufacture of a vaccine.
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, the glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein, for the treatment of a subject having a disease associated with increased expression of said one or more carbohydrate or sugar antigens.
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, the glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein, for producing an antibody that specifically binds to the glycoconjugate immunogen.
  • the present description relates to the use of the glycoconjugate immunogen prepared by a method as described herein, the glycoconjugate vaccine produced by a method as described herein, a synthetic glycoconjugate immunogen as described herein, or a vaccine as described herein, for detecting an antibody that specifically binds to the glycoconjugate immunogen.
  • the present description relates to a method for producing a glycoconjugate, the method comprising: (a) providing a carbohydrate antigen covalently linked to a terminal alkene (alkenyl carbohydrate antigen), the terminal alkene being directly conjugatable to a thiol group via a thiol-ene reaction; (b) providing a conjugate material suitable for conjugation to the carbohydrate antigen via a thiol-ene reaction as described herein, said conjugate material having one or more free thiol groups; and (c) performing a photocatalytic thiol-ene reaction to directly conjugate the carbohydrate antigen to the conjugate material at the one or more free thiol groups, thereby producing the glycoconjugate.
  • the conjugate material is or comprises a polymer, a polypeptide, a carrier protein as defined herein, a solid support, a particle, or any other material having a free thiol group suitable for conjugation to the carbohydrate antigen via a thiol-ene reaction as described herein.
  • the present description relates to the use of a glycoconjugate produced by a method described herein, for detecting or screening for the presence of an antibody that specifically binds to a carbohydrate antigen or a tumor-circulating cell comprising a carbohydrate antigen, or for detecting the presence of antibodies resulting from an immunization or vaccination with a carbohydrate antigen.
  • the detection or screening may be performed via any suitable detection method, such as an immunosorbent assay, ELISA, microarray, or immunoblot analysis.
  • the present description relates a method of treating a subject comprising administering a glycoconjugate or glycoconjugate immunogen as defined herein or produced by a method as described herein, to generate an immune response in said subject to a carbohydrate antigen, and screening a biological sample from said subject for the presence of antibodies that specifically binds to the carbohydrate antigen.
  • a synthetic glycoconjugate immunogen comprising one or more sugar antigens covalently linked to one or more free thiol groups of a carrier protein via a linker, wherein the synthetic glycoconjugate immunogen has the structure:
  • UV-AC Hand Lamp Dual 254/365 nm UV; 115V-60 Hz, 0.16 amps, VWR Canada, Cat. No. 89131-492.
  • Flash chromatography was performed using ZEOprepTM silica gel 60 (40-63 ⁇ m) from Canadian Life Science. Detection was carried out under UV light or by spraying with 20% ethanolic sulfuric acid or molybdate or KMnO 4 solution followed by heating. NMR spectra were recorded on Bruker ULTRASHIELDTM 300 MHz and Bruker AvanceTM III HD 600 MHz spectrometers.
  • High-resolution mass spectra were measured with a LC-MS-TOF (Liquid Chromatography Mass Spectrometry Time Of Flight) instrument from Agilent technologies in positive and/or negative electrospray mode by the analytical platform of UQAM. Either protonated ions (M+H) + or sodium adducts (M+Na) + were used for empirical formula confirmation.
  • the native TT and TT-conjugate were dialyzed using 2000 KDa benzoylated dialysis tubing (Sigma-Aldrich (Ontario, Canada). The thiol contents of both native and conjugated TT were determined by the Ellman test at 412 nm (Ellman, G. L. Arch. Biochem.
  • the total sugar content of the TT-conjugate was determined by the colorimetric DuBois test measured at 492 nm (Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem., 1956, 28, 350-356) using a UV/VIS Ultrospec 100 prot spectrophotometer (Biochrom, USA). Dynamic Light Scattering (DLS), particle size distributions were measured in PBS using a Zetasizer Nano S90 from Malvern. The mouse monoclonal IgG3 antibody JAA-F11 was produced as previously described in Rittenhouse-Diakun et al., 1998.
  • SPPS General Solid Phase Peptide Synthesis
  • the Fmoc-protecting group of the commercial resin or of amino acids were removed with a solution of 20% piperidine in DMF (5 mL, 2 ⁇ 5 min then 1 ⁇ 10 min). The solvents and reagents were removed by filtration, and the resin was washed with DMF, CH 2 Cl 2 and MeOH (3 ⁇ with each solvent). The presence of free amino groups was verified by a Kaiser test or TNBS test.
  • Acetyl chloride (2.76 mL, 38.80 mmol, 3.43 equiv.) was added dropwise to allylic alcohol (20.8 mL) at 0° C. under argon atmosphere.
  • N-acetyl-D-glucosamine (Compound 1) (2.50 g, 11.3 mmol, 1.00 eq.) was added.
  • the reaction mixture was stirred at 70° C. for 3 hours, then quenched by adding solid NaHCO 3 until pH 7.
  • the suspension was filtered through out a pad of Celite, washing several times with MeOH.
  • FIGS. 6C & 6D ESI + -HRMS: [M+H] + calcd for C 21 H 36 O 8 N, 430.2435. found, 430.2445.
  • the ⁇ -anomer was isolated as white solid (971 mg, 2.26 mmol, 20%).
  • di-O-pivaloyl compound (Compound 2) (5.50 g, 12.80 mmol, 1.0 equiv.) in a mixture of dry dichloromethane-pyridine (126 mL, v/v 20:1) was cooled to ⁇ 35° C. under argon atmosphere. Trifluoromethanesulfonic anhydride (2.58 mL, 15.36 mmol, 1.2 equiv.) was then added and the mixture was stirred at this temperature. The temperature was warned to room temperature for 2 hours. Water (12 mL) was then added into the solution. The mixture was heated, and stirred at reflux 50° C.) overnight (12 hours).
  • the reaction mixture was diluted with dichloromethane and washed with 1M aqueous HCl several times. The organic layer was washed with H 2 O, saturated NaHCO 3 , and brine. The organic layer was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure.
  • the crude product was treated under Zemplén condition (1M sodium methoxide solution in methanol, 40 mL, pH 9). The solution was stirred at 50° C. overnight. After cooling to room temperature, the solution was neutralized by addition on ion-exchange resin (Amberlite® IR 120H + ), filtered, washed with MeOH, and the solvent was removed under reduced pressure.
  • FIGS. 7C & 7D ESI + -HRMS: [M+H] + calcd for C 11 H 20 O 6 N, 262.1285. found, 262.1294.
  • Allyl 2-acetamido-2-deoxy- ⁇ -D-galactopyranoside can also be directly prepared from N-acetylgalactosamine (GalNAc) according to literature procedure (Feng et al., 2004: To a solution of N-acetylgalactosamine (442 mg, 2 mmol, 1.0 equiv.) in allyl alcohol (8 mL) at room temperature was added BF 3 .Et 2 O (250 ⁇ L, 2 mmol, 1.0 equiv.), and the mixture was stirred at 70° C. for 2 hours. The solution was cooled to room temperature and the solvent was removed under reduced pressure. The dry crude product was dissolved in minimum EtOH (5 mL). The desire allyl T N product was precipitated in diisopropyl ether and isolated as white solid (417 mg, 1.60 mmol, 80%).
  • the C-Allyl GalNAc analog [1-(2′-Acetamido-2′-deoxy- ⁇ -D-galactopyranosyl)-2-propene] has been prepared according to literature procedure (Cipolla, et al., 2000: 3-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy- ⁇ -D-galactopyranosyl)-1-propene (Cui et al., 1998) (371 mg, 1.00 mmol, 1.0 equiv.) was treated under Zemplén condition (1M sodium methoxide solution in methanol, 5 mL, pH 8-9). The solution was stirred at room temperature for 1 h.
  • the S-Allyl GalNAc analog ( FIG. 5 ) was prepared according to literature: Knapp et al., 2002.
  • FIGS. 10C & 10D ESI + -HRMS: [M+H] + calcd for C 24 H 34 O 11 N, 512.2126. found, 512.2119.
  • Tetanus toxoid (TT) monomer was obtained by gel filtration chromatography before conjugation.
  • One milliliter of a liquid preparation containing 4.5 mg/ml protein (as determined by the modified Lowry protein assay) was loaded onto a XK16-100 column filled with Superdex®200 Prep Grade (GE Healthcare Life Sciences, Uppsala, Sweden) equilibrated in PBS (20 mM NaHPO 4 [pH 7.2], 150 mM NaCl) and eluted with the same buffer.
  • Fractions corresponding to the later (monomer) peak were pooled, desalted against deionized water, concentrated using a Centricon® Plus-70 centrifugal filter device (30K Ultracel PL membrane; Millipore, Billerica, Mass.), and then lyophilized.
  • Procedure B Under the same thiol-ene click reaction, a catalytic amount of DMPA in acetonitrile (100 ⁇ L) was added into the solution of allyl Tn (3 mg, 10.8 ⁇ mol, 180.0 equiv.) containing Tetanus toxoid in PBS (2 mL, 4.5 mg/mL, 0.06 ⁇ mol). The solution was irradiated at 365 nm for 15 min. The solution was then dialyzed for 24 hours, and lyophilized to give Tn-TT-B conjugate as white solid (5.4 mg, 61%).
  • Procedure B Under the same thiol-ene click reaction, a catalytic amount of DMPA in acetonitrile (100 ⁇ L) was added into the solution of allyl TF (5 mg, 10.8 ⁇ mol, 180.0 equiv.) with Tetanus toxoid in PBS (2 mL, 4.5 mg/mL, 0.06 ⁇ mol). The solution was irradiated at 365 nm for 15 min. The solution was then dialyzed for 24 hours, and lyophilized to give TF-TT-B conjugate as white solid (5.3 mg, 60%).
  • tetanus toxoid alone and the vaccine conjugates (Tn-TT-A; Tn-TT-B; TF-TT-A; and TF-TT-B) were analyzed by SDS gel electrophoresis, colorimetric analyses for the presence of sugar content (Dubois test), thiol content by the Ellmann test, Dynamic Light Scattering (DLS) ( FIG. 12 ), and reactivity with the known mouse monoclonal antibody JAA-F11 using double radial immunodiffusion.
  • the SDS gel electrophoresis results clearly indicated the monomeric form of the conjugate that also stained positive for the presence of the carbohydrate antigens (Tn and TF).
  • the colorimetric analyses confirmed the presence of the carbohydrate antigens (10% by weight) and that there were no residual free thiol groups on the carrier protein following conjugation.
  • the double radial immunodiffusion analyses revealed a precipitation band, clearly indicating the cross-reactivity of the new TF-TT conjugate with the anti-TF monoclonal antibody JAA-F11, thus confirming the presence of the immunogenic carbohydrate antigen (TF) on the TF-TT conjugate and that no precipitation band was observed with the carrier protein (tetanus toxoid) alone.
  • HPLC analysis of the glycoconjugate preparations was done by size exclusion chromatography. The chromatographic separation was performed with three 8-by 300-mm Shodex OHpak gel filtration columns connected in series (two SB-804 and one SB-803) preceded by a SB-807G guard column (Showa Denko).
  • the glycoconjugate immunogens were eluted with 0.1 M NaNO 3 at a flow rate of 0.4 mL/min using a Knauer Smartline system equipped with a differential refractometer (RI) detector model 2300 and a UV detector model 2600 at wavelength of 280 nm.
  • the conjugate preparation (8-mg/mL solution in the mobile phase) was injected using a 504 ⁇ injection loop.
  • the fractions eluting at the void volume which correspond to the conjugate fractions, were pooled, dialyzed against water Spectra/Por; Molecular weight cut-off (MWCO), 12,000 to 14,000 [Spectrum Laboratories]), and lyophilized. This corresponds to the 2:1 fractionated conjugate.
  • the peptide dTT831-844-Cys- ⁇ Ala (Tetanus Toxin (831-844); MW: 1813, SEQ ID NO: 2) was solubilized in water at a concentration of 3.7e-3 M and (500 uL) of peptide was stirred at room temperature with (6 uL, 0.1M, 3.0 equiv.) O-Allyl-Tn or O-Allyl-TF and water-soluble catalyst AAPH (2,2′-Azobis(2-methylpropionitrile, 23 uL, 0.025M, 3.0 equiv.
  • FIG. 13 shows that the free thiol concentration is reduced in a time dependent manner as a result of the photo thiol-ene reaction.
  • FIG. 14 presents the fold reduction in free thiol concentration for dTT831-844 peptides having an N-terminal (“SH-peptide”) or C-terminal (“Peptide-SH”) cysteine, and using either a water-soluble (AAPH) or water-insoluble activator (DMPA), in the thiol-ene photoreaction conjugation.
  • SH-peptide N-terminal
  • Peptide-SH C-terminal cysteine
  • the immunoreactivity of the thiol-ene photoreaction product was measured by an Enzyme-linked lectin assay (ELLA). 1 ⁇ g of peptide in 100 ⁇ L PBS pH 7.4 was allowed to adsorb to 96-well plates (MaxisorpTM, Nunc) for a minimum of 1 h at RT or overnight at 4° C. The wells were then washed twice with PBS-TweenTM 0.05% (PBS-T) and then filled with PBS-T+1% BSA blocking solution for 30 minutes.
  • ELLA Enzyme-linked lectin assay
  • FIG. 15 presents lectin reactivity to the different Tn-peptides (i.e., unconjugated: “pep-Cys” and “Cys-pep”; Tn-conjugated: “Tn-pep” and “pep-Tn”; “unc”: uncoated plate) using either a water-soluble (AAPH) or water-insoluble activator (DMPA).
  • AAPH water-soluble
  • DMPA water-insoluble activator
  • TT peptides with a C-terminal cysteine were specifically recognized by the anti-Tn lectin VVA, and the conjugation reaction was most effective with the water-soluble activator AAPH. None of the Tn glycoconjugates were recognized by the anti-TF lectin (peanut lectin, PNA) used as a negative control.
  • the peptide dTT831-844-Cys- ⁇ Ala (MW: 1813, SEQ ID NO: 2) was stirred at room temperature with O-Allyl-Tn 0.55 mM in 1 mL water and AAPH (1.44 mg, 5.5 nmol, 10.0 equiv.) or (0.3 mg, 1.1 nmol, 2.0 equiv.) DMPA in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at either 365 nm or 355 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH and turned off to stop the reaction after (60 min).
  • the peptide was also conjugated to O-Allyl-Tn in the absence of activator and at UV254 nm.
  • the free thiol concentration was measured by the Ellman test using a cysteine standard curve.
  • FIG. 16 shows the decrease in free thiol over time (minutes) for three different conjugation conditions (“355 nm+AAPH”; “365 nm+DMPA”; and “254 nm”).
  • the immunoreactivity of the thiol-ene photoreaction product was measured by Enzyme-linked lectin assay (ELLA).
  • the indicated quantity of peptide in 100 ⁇ L PBS pH 7.4 was allowed to adsorb to 96-well plates (Maxisorp, Nunc) for a minimum of 1 h at RT or overnight at 4° C.
  • the wells were then washed twice with PBS-TweenTM 0.05% (PBS-T) and the wells filled with blocking solution for 30 minutes.
  • the wells were then washed 3 times with PBS-T and filled with 100 ⁇ L of a 1/100 dilution of the lectin Vicia Villosa coupled to the horseradish peroxidase (VVA-hrp, EY LAbs). After an incubation of 1 h at RT with gentle shaking or 12 h at 4° C., the wells were washed 4 times with PBS-T and 100 ⁇ L of hrp substrate was added (Ultra TMB-ELISA, Thermo Scientific) to each well. The reaction was stopped by the addition of 100 ⁇ L of 0.5N sulfuric acid and the plate was read in a plate reader at 450 nm (Biotek EL808).
  • FIG. 17 shows VVA lectin reactivity to various quantities of Tn-peptides conjugated by thiol-ene photoreaction at 365 nm in the presence of AAPH or DMPAP, or at 254 nm in absence of activator.
  • the conjugation products were detected by the lectin, while the unconjugated peptide (“peptide”) or O-Allyl-Tn alone (“Tn”) were unreactive.
  • FIG. 17 shows that conjugation at 254 nm in the absence of activator generated a glycoconjugate product that was more immunoreactive than the ones generated by 365 nm or 355 nm in the presence of activator.
  • the dTT831-844-Cys- ⁇ Ala peptide (MW: 1813) was solubilized in water at a concentration of 0.55 mM and 1 mL of it was stirred at room temperature with O-Allyl-Tn, O-Allyl-TF, or C-Allyl-Tn (100 uL, 11 mM, 2.0 equiv) and AAPH (1.44 mg, 5.5 nmol, 10.0 equiv.) in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH.
  • FIG. 18 shows the decrease in free thiol over time (minutes) for the different allyl saccharides tested: O-Allyl-Tn (“Tn”), O-Allyl-TF (“TF”), or C-Allyl-Tn (“cTn”).
  • the immunoreactivity of the thiol-ene photoreaction product was measured by enzyme-linked lectin assay and enzyme-linked immunosorbent assay.
  • the indicated quantity of peptide conjugate in 100 ⁇ L PBS pH 7.4 was allowed to adsorb to 96-well plates (Maxisorp, Nunc) for a minimum of 1 h at RT or overnight at 4° C.
  • the wells were then washed twice with PBS-TweenTM 0.05% (PBS-T) and then filled with PBS-T+1% BSA blocking solution for 30 minutes.
  • FIG. 19 shows that only the peptide conjugate generated with the O-Allyl-Tn (but not with the C-Allyl-Tn) was recognized by the anti-Tn lectin VVA.
  • Detoxified tetanus toxoid was dialysed in PBS pH 7.4 (MWCO 2,000) then incubated with 1000 eq DTT. After a 2 hours incubation at RT and rotation, the reduced protein solution was washed in PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon).
  • the reduced protein (1 mL, 3.51 mg/mL, 0.02 nmol) was then stirred with O-Allyl-TF (30.0 equiv.) and AAPH (2.0 equiv.) in a final volume of 1.0 mL PBS in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction then stopped after 2 h.
  • the free thiol concentration of the reduced dTT generated by the DTT treatment compared to native dTT was measured on buffer exchanged dTT sample by an Ellman test using a cysteine standard curve and the protein concentration was measured by a Bradford assay using a BSA standard curve.
  • the plate was washed with PBS-T then further incubated for 60 minutes with 100 ⁇ L of 1/1000 dilution of the secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch). Wells were then washed 4 times with PBS-T and 100 ⁇ L of hrp substrate was added (Ultra TMB-ELISA, Thermo Scientific) to each well. The reaction was stopped by the addition of 100 ⁇ L of 0.5N sulfuric acid and the plate was read in a plate reader at 450 nm (Biotek EL808).
  • FIG. 20 shows the immunoreactivity dTT-TF to JAAF11 (“dTT(DTT)-TF”), while the reduced dTT (“dTT(DTT)”) or native dTT (data not shown) were unreactive. This indicates that the O-Allyl-TF was indeed conjugated to dTT by the thiol-ene photo reaction in an immunoreactive conformation.
  • dTT was dialysed in PBS pH 7.4 (MWCO 30,000) then 0.5 mL (5.8 mg/mL) was incubated with 50 or 500 eq DTT at RT under rotation. After 2 h, the reduced protein solution was washed in PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon).
  • the protein reduced with 500 eq of DTT (70 uL, 0.5 mmol/mL) was then stirred with 0.1M O-Allyl-TF (60.0 equiv.) and 0.1M AAPH (4.0 equiv.) in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette.
  • the UV was turned on immediately following the addition of AAPH to initiate the reaction then stopped after 45 minutes.
  • the conjugated product was treated with 100 Eq of DTT for 1 h then washed by centrifugal filtration, and then the thiol content was measured before and after the reducing treatment.
  • the free thiol dosage was measured by an Ellman test using a cysteine standard curve.
  • the protein concentration was measured by a Bradford assay using a BSA standard curve.
  • FIG. 21 shows the molar ratio of free thiols in dTT after the different conjugation conditions.
  • the results indicate that the dTT protein was reduced in a dose-dependent manner and reached 9 free thiols upon treatment with 500 Eq DTT (out of a maximal theoretical number of 10 cysteines in dTT).
  • Conjugation of O-Allyl-Tn or O-Allyl-TF by the thiol-ene photo-reaction lowered the free thiol to at or below the level of the non-reduced dTT.
  • the resistance of the dTT-Tn conjugate to reduction confirmed the presence of a reduction-resistant thio-ether bond that formed by the thiol-ene photo reaction.
  • the wells with JAAF11 were washed with PBS-T then further incubated for 60 minutes with 100 ⁇ L of 1/1000 dilution of the secondary antibody goat anti-mouse IgG (H+L)-hrp (Jackson Immunoresearch).
  • the plate washed 4 times with PBS-T and 100 ⁇ L of hrp substrate was added (Ultra TMB-ELISA, Thermo Scientific) to each well. The reaction was stopped by the addition of 100 ⁇ L of 0.5N sulfuric acid and the plate was read in a plate reader at 450 nm (Biotek EL808).
  • FIG. 22 shows the high reactivity of the dTT-Tn conjugate to the lectin VVA compared to the unconjugated dTT.
  • the native dTT and dTT-Tn conjugate were analyzed by SDS-PAGE electrophoresis on a 10% polyacrylamide gel, then stained with Coomassie blue. The gel electrophoresis results showed that the dTT-Tn conjugate migrated at higher molecular weight than the native non-conjugated dTT (data not shown).
  • dTT was dialysed in PBS pH 8 (MWCO 30,000) then 100 ⁇ L (0.8 mg) was stirred with 200 Eq/protein of a solution of 0.1 M 2-imminothiolane hydrochloride in water (Sigma), 200 Eq/protein of 0.1 M O-Allyl-TF in water, and 0.8 Eq/protein or approximately 450 Eq/protein AAPH in water in a final volume of 124 ⁇ L in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction.
  • FIG. 23 shows that the ratio of free thiol in the dTT samples subsequent to chemical thiolation with 2-imminothiolane and thiol-ene photoreaction in the absence of O-Allyl-Tn reaches 25-30, while the presence of O-Allyl-Tn reduces the thiol ratio of dTT to approximately 20 and 5 in a dose-dependent manner respective to the AAPH concentration, suggesting that a thio-ether bond was formed by the thiol-ene photo-reaction.
  • the plate washed 4 times with PBS-T and 100 ⁇ L of hrp substrate was added (Ultra TMB-ELISA, Thermo Scientific) to each well. The reaction was stopped by the addition of 100 ⁇ L of 0.5N sulfuric acid and the plate was read in a plate reader at 450 nm (Biotek EL808).
  • FIG. 24 shows the reactivity of dTT samples to the lectin VVA-hrp. Higher reactivity of Tn conjugates compared to native unconjugated dTT was observed. Further, the reactivity correlated with the duration of the photo-reaction and the amount of AAPH activator used.
  • the dTT conjugates (5 ⁇ g) were analyzed by SDS-PAGE electrophoresis on a 10% polyacrylamide gel either stained with Coomassie blue or transferred to a membrane (Immobilon-P, Merck Millipore) for western blot analysis.
  • the membrane was blocked in PBS-T+1% BSA for 1 h, washed with PBS-T, then incubated with a 1/100 dilution of the lectin Vicia Villosa coupled to the horseradish peroxidase (VVA-hrp, EY LAbs) in PBS-T for 1 h at RT or overnight at 4° C.
  • the membrane was then extensively washed with PBS-T and the bound VVA-hrp was detected with the horseradish peroxidase chromogenic substrate 4-Chloro 1-Naphthol (0.3 mg/mL in PBS containing 0.03% hydrogen peroxide, Sigma).
  • BSA (7 mg in 1 mL PBS, pH 7.4) was incubated with 600 eq DTT for 2 h then washed in PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon). The reduced protein was then conjugated to O-Allyl-Tn or to O-Allyl-TF by the thiol-ene photoreaction.
  • Reduced BSA 600 uL, 5.3 mg/mL, 0.047 nmol was stirred with of O-Allyl-sugar (60 eq) and AAPH (4.0 equiv.) and PBS pH 7.4 (720 uL) in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction. After 45 min., the product was washed with PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon) to remove any un-reacted O-Allyl and reagents. The free thiol concentration of the reduced BSA was measured by Ellman assay using a cysteine standard curve, and the protein concentration by a Bradford assay using BSA as standard.
  • FIG. 25 shows that the BSA-TF and BSA-Tn generated under these conditions were highly reactive and specific to anti-TF JAAF11 monoclonal antibody and for the anti-Tn lectin VVA, respectively.
  • the BSA-Tn and BSA-TF conjugates (5 ⁇ g) were analyzed by SDS-PAGE electrophoresis on a 10% polyacrylamide gel stained with Coomassie blue.
  • the reduction of BSA under the conditions used generated a molar ratio of 19.29 thiols as measured by the Ellman test.
  • the Ellman's test was negative, suggesting that the free thiols were chemically coupled to the O-Allyl sugars by the thiol-ene photoreaction (data not shown).
  • the Coomassie-stained SDS-PAGE gel showed both BSA-conjugates migrated at the expected molecular weight of BSA, and to higher molecular weight species (data not shown).
  • BSA (500 ⁇ L, 7 mg/mL) was incubated with 400, 200 or 1000 Eq of DTT (0.5 M) for 1 h at RT and rotation.
  • the reduced BSA solution was then dialysed overnight in PBS pH 7.4.
  • the free thiol concentration of the reduced BSA was then measured by Ellman assay using a cysteine standard curve, and the protein concentration by a Bradford assay using BSA as standard, from which the molar ratio of free thiol/BSA was calculated.
  • FIG. 26 shows a correlation between the amount of free thiol generated and the amount of the reducing agent DTT, which reaches about 10 thiol/BSA when the reduction was conducted with 1000 Eq DTT under the conditions employed.
  • the reduced protein was then conjugated to various ratios of O-Allyl-Tn.
  • Reduced BSA (5 nmols) was stirred with 100 Eq of O-Allyl-Tn/reduced BSA or 100 Eq of O-Allyl-Tn/BSA-thiol with AAPH and PBS pH 7.4 in a final volume of 500 ⁇ L in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction. After 45 minutes, the product was washed with PBS pH 7.4 by centrifugal filtration (MWCO 10,000, Amicon) to remove unreacted 0-Allyl and reagents.
  • MWCO 10,000, Amicon centrifugal filtration
  • the wells were then washed 3 times with PBS-T and filled with 100 ⁇ L of a 1/100 dilution of the lectin Vicia Villosa , coupled to the horseradish peroxidase (VVA-hrp, EY LAbs) or 0.1 ⁇ g/mL of TF-specific purified murine monoclonal antibody JAAF11.
  • VVA-hrp horseradish peroxidase
  • EY LAbs horseradish peroxidase
  • JAAF11 horseradish peroxidase
  • FIG. 27 shows the reactivity of the various BSA-Tn conjugates to the lectin VVA and to the anti-TF monoclonal antibody JAAF11 (+goat anti-mouse IgG-hrp conjugate).
  • the BSA-Tn conjugates (5 ⁇ g) were analyzed by SDS-PAGE electrophoresis on a 10% polyacrylamide gel either stained with Coomassie blue or for glycoproteins according to the manufacturer's instructions (Pierce). The results showed that the BSA conjugates that migrated at a high molecular weight were positively stained for glycan, while the native unconjugated BSA was not (data not shown).
  • BSA (1 mL, 0.088 ⁇ mol, 5.89 mg/mL, PBS pH 8.0) was incubated with 5, 10, or 20 Eq of 2-imminothiolane (5, 10, 20 ⁇ L, 0.1 mmol/mL) for 1 h. Thiolated BSA was then washed by centrifugal filtration (MWCO 10,000, Amicon) to remove unreacted reagents before conjugating to O-Allyl-TF by thiol-ene photoconjugation.
  • MWCO 10,000, Amicon centrifugal filtration
  • Thiolated BSA (440; 480; 575 ⁇ L, 6.8; 6.4; 5.2 mg/mL) was stirred with 3 eq/thiol of O-Allyl-Tn and AAPH (0.2 Eq/thiol) and PBS pH 7.4 in a final volume of 1 mL in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction.
  • the wells were then washed 3 times with PBS-T and filled with 100 ⁇ L of a 1/100 dilution of the lectin peanut agglutinin coupled to the horseradish peroxidase (PNA-hrp, EY LAbs), or 0.1 ⁇ g/mL of TF-specific purified murine monoclonal antibody JAAF11.
  • PNA-hrp horseradish peroxidase
  • EY LAbs horseradish peroxidase
  • JAAF11 horseradish peroxidase
  • the dual axis graph shows the reactivity of the BSA-TF conjugates to the anti-TF lectin PNA or mAb JAAF11 in relation to the thiol ratio of the respective conjugates measured before TF conjugation.
  • BSA 250 ⁇ L, 4.3 mg/mL
  • 200 eq of 0.1 M 2-imminothiolane Sigma
  • 200 Eq of 0.1 M O-Allyl-Tn approximately 450 Eq AAPH and PBS pH 8
  • a quartz cuvette (10 ⁇ 10 mm path length, Fisher) placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette.
  • the UV was turned on immediately following the addition of AAPH to initiate the reaction. Samples were taken at the indicated time during the reaction.
  • the reaction was stopped after 45 minutes after which the product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using SephadexTM G25 mini spin column.
  • the free thiol concentration of the BSA samples were measured by Ellman assay using a cysteine standard curve, and the protein concentration by a Bradford assay using BSA as standard.
  • FIG. 29 shows a time-dependent increase of free thiol function upon reaction of the BSA with 2-imminothiolane alone or in the presence of 365 nm light, while the addition of AAPH in the presence or the absence of O-Allyl-Tn in the photoreaction prevent the formation of detectable free thiols.
  • the reactivity of the BSA conjugates to the lectin VVA indicates that only the BSA generated by the thiol-ene photoreaction in the presence of O-Allyl-Tn is detected by the VVA, while the product generated in the absence of O-Allyl-Tn is negative in the ELLA.
  • BSA 250 ⁇ L, 4.3 mg/mL was incubated with 200 Eq of 0.1 M 2-imminothiolane (Sigma), 200 Eq of 0.1 M O-Allyl-Tn, and either 0.8 Eq of AAPH or approximately 450 Eq AAPH and PBS pH 8 in a final volume of 316 ⁇ L in a quartz cuvette (10 ⁇ 10 mm path length, Fisher).
  • the effect of the anti-oxidant Vitamin C (approx. 5 mM) on conjugation was tested in the condition containing a high amount of AAPH.
  • the cuvettes were placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette.
  • the UV was turned on immediately following the addition of AAPH to initiate the reaction. Samples were taken at the indicated time during the reaction. The reaction was stopped after 45 minutes after which the product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using SephadexTM G25 mini spin column. The free thiol concentration of the conjugated BSA samples was measured by Ellman assay using a cysteine standard curve, and the protein concentration by a Bradford assay using BSA as standard.
  • FIG. 31 having a dual Y-axis shows an increase in reactivity of the conjugation product to the lectin VVA as a function of time, and a corresponding decrease in free-thiol as a result of the thiol-ene photoconjugation of the O-Allyl-Tn to BSA.
  • the BSA-Tn conjugates (5 ⁇ g) were analyzed by SDS-PAGE electrophoresis on a 10% polyacrylamide gel stained with Coomassie blue. The results showed a higher amount of high molecular weight species in the reaction at high AAPH than at lower AAPH. Vitamin C prevented the formation of high molecular weight species (data not shown).
  • BSA (93 ⁇ L, 11.7 mg/mL) was incubated with 200 Eq of 0.1 M 2-imminothiolane (Sigma), 200 Eq of 0.1 M O-Allyl-Tn and either 0.08 Eq, 0.8 Eq or approximately 450 Eq AAPH and PBS pH 8 in a final volume of 316 ⁇ L in a quartz cuvette (10 ⁇ 10 mm path length, Fisher).
  • the 2-imminothiolane and/or the AAPH was omitted and replaced by PBS pH 8.
  • the cuvette was placed between two hand held UV lamps at 365 nm or at 254 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette.
  • the UV was turned on immediately following the addition of AAPH to initiate the reaction.
  • the reaction was stopped after 45 minutes after which the product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using SephadexTM G25 mini spin column.
  • the protein concentration was measured by a Bradford assay using BSA as standard.
  • FIG. 32 shows the minimal reactivity of product to VVA when the conjugation is performed in absence of protein thiolation or when the thiolated protein is conjugated in the absence of activator AAPH.
  • a VVA reactive product is generated by UV254 nm in the absence of protein thiolation and AAPH.
  • BSA (93 ⁇ L, 11.7 mg/mL) was incubated in the presence or absence of 200 Eq of 0.1 M 2-imminothiolane (Sigma), 200 Eq of 0.1 M O-Allyl-TF and 0.8 Eq or approximately 450 Eq AAPH and PBS pH 8 in a final volume of 316 ⁇ L in a quartz cuvette (10 ⁇ 10 mm path length, Fisher) on the bench at RT or placed between two hand held UV lamps at 365 nm (0.16 amps, VWR) at a distance of 2-5 cm from the cuvette. The UV was turned on immediately following the addition of AAPH to initiate the reaction.
  • the reaction was stopped after 45 minutes after which the product was buffer exchanged to PBS pH 7.4 by gel filtration chromatography using SephadexTM G25 mini spin column.
  • the protein concentration was measured by a Bradford assay using BSA as a standard.
  • the conjugated galactose of the TF epitope was measured by the method of Dubois using a galactose standard.
  • FIG. 33 having a dual Y-axis shows the lower immunoreactivity of the BSA-TF with a high galactose ratio compared to the lower galactose conjugate.
  • the detection of galactose in the two conjugates confirms that TF is conjugated to BSA but conjugated TF immunoreactivity is compromised at high ratio.
  • FIG. 34 shows the chemical structures of the dTT831-844-Cys- ⁇ Ala and the Cys-dTT831-844- ⁇ Ala with a cysteine residue at the C- and N-terminal, respectively together with their tabulated LC-MS data.
  • FIG. 35 shows the detailed LC-MS data of the peptide dTT831-844-Cys- ⁇ Ala (C-terminal).
  • FIG. 36 shows the detailed LC-MS data of the peptide Cys-dTT831-844- ⁇ Ala (N-terminal).
  • FIG. 37 shows the photolytic AAPH-catalyzed thiol-ene reaction of the O-Allyl Tn on the C-terminal peptide dTT831-844-Cys- ⁇ Ala.
  • FIG. 38 shows the LC-MS profile of the O-Allyl Tn on the C-terminal peptide
  • FIG. 39 shows the photolytic AAPH-catalyzed thiol-ene reaction of the O-Allyl Tn on the N-terminal peptide Cys-dTT831-844- ⁇ Ala.
  • FIG. 40 shows the detailed LC-MS profile of the O-Allyl Tn on the N-terminal peptide.

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